Process for culturing organisms



Jan. 29, 1963 a. B. ACHORN, JR; ETAL 3,

PROCESS FOR CULTURING ORGANISMS Filed Sept. 1:. 1961 Fig./

IN VE/V TORS Elmer R. fiokesc/r Edward R. Dapper Robert-W Labherz Samuel IV. Mefcalfe Arthur Rawson John L. Schwab James R. E. Smith QMM ATTORNEY Glenwaad B. Achorn Jr:

Patented Jan. 29, 19E

3,075,888 PROCESS FOR CULTURING ORGANISMS Glenwood B. Achorn, Jr., P.0. Box 3204, Arsenal, Ark.; Elmer R. Bokesch, 17 Kline Blvd., Frederick, Md.; Edward R. Dapper, Rte. 3, Frederick, Md.; Robert W. Lebherz, Jr., Rte. 5, Frederick, Md.; John L. Schwab, 102 3 Fox Chase Road, Birmingham, Mich.; James R. E. Smith, 506 W. Patrick St., Frederick, Md.; Arthur J. Rawson, Rte. 7, Frederick, Md.; and Samuel N. Metcalie, In, New Windsor, Md.

Filed Sept. 13, 1961, Ser. No. 137,927 4 Claims. (Cl. 195-127) (Granted under Title 35, U.S. Code (1952), see. 266) The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment to us of any royalty thereon.

This is a subcombination of our invention covered in Patent No. 2,981,660, issued April 25, 1961.

This invention relates to the cultivation of microorganisms. It relates more particularly to the apparatus and method for the safe and efficient production of pure cultures of pathogenic fastidious microorganisms by the fermentation process in submerged cultures. Further, it relates to a novel drain valve, a novel aeration device and a unique process of aeration and agitation.

Past experience in the design of equipment for producing pure cultures of-fastidious organisms has demonstrated the need for marked departures from the techniques commonly employed by the fermentation industry. Designs similar to those used for production of alcohol and antibiotics were tested and found to be unsuitable. Over 50 percent of the cultures of fastidious microorganisms prepared in this type of equipment became contaminated to a point where the data obtained could not be considered reliable.

The results of investigation into the causes for contamination indicated that the systems could not always be sterilized with either liquid decontaminants or steam at a pressure of 40 pounds per square inch gauge. The primary sources of contamination, after attempted sterilization, were valves, pipe ends, pockets or crevices, packing glands, and flanges. Also, the isolation of the fermentor unit was found necessary in order to prevent cross-contamination between units. Therefore, it is recognized that there is a need for unique equipment that will provide the isolated system in which fastidious, pathogenic microorganisms can be developed by particular fermentation processes.

Furthermore, there is also a requirement of efficient growth conditions for the microorganisms. Among these conditions, eflicient aeration has been known to be a large factor in microorganism growth, but present methods and apparatus have left something to be desired in the achievement of desirable aeration efliciencics, while maintaining an isolated system.

Hence, the primary objects of this invention are two in number. The first primary object is to provide apparatus and means for safely producing pure cultures of fastidious microorganisms without danger of the con tamination of the environment or of the organism.

The second primary object is to provide a novel apparatus and method for efiicient aeration.

A further object is to provide a novel drain valve.

Another object is to provide a novel drain valve in a tank that assures against loss of the contents of the tank.

Another object is to provide novel means for sampling the contents of a tank.

Further objects, as well as a clear understanding of the present invention, will be had by reference to the following detailed description and drawings.

FIG. 1 is a diagrammatic view in front elevation the system illustrating the component parts of the s tern and the piping arrangement by which the compone: are integrated in the system.

FIG. 2 is a partial, sectionalized view of the aerati device shown in relation to its position with resp to the wall of the fermentor vessel and the source of supply.

FIG. 3 is a sectional view of the sampling adap assembly.

FIG. 4 is a partial sectional view in elevation of 1 novel drain valve.

Referring now to FIG.- 1, the fermentation tank fermentor 10 is an all welded vessel preferably made stabilized stainless steel described in Stainless Steel Hai book, Allegheny Ludlum Steel Corporation, Pittsbur; Pennsylvania, 1956. All welds in the tank are grou smooth and surfaces of the fermentor 10 which are come into contact with the culture are polished, e.g., it number 4 finish, ASTM standard, in order to facilit: cleaning.

Through experience, it has been learned that a pro: nent cause of contamination in fermentation proces has been a lack of complete sterilization of the fermen tion tank. A cause of this incomplete sterilization l been the inability of a decontaminating material to t contaminate foreign matter from in and around varit burrs, projections and crevices in the metal surf: of the tank. Thus, a removal of these imperfections a polishing the surface of the tank eliminates one of 1 major problems in sterilization' of fermentation tanks.

The tank 10 has disposed in its upper portion a grou faced media inlet 15 with valve 14 adapted to rece the corresponding end of a media line 12, a centra disposed gas removal or steam purging line 16, optiona fluidly connected to steam line 19 by valve 18, but constant fluidal communication with gas exit line 17 cated as shown in FIG. 1. Also in the upper portion the tank is situated a sampling adaptor 20 similar to l sampling adaptor 22 positioned in the lower portion the tank, the structure of which will be set forth in tail infra.

In the lower portion of the tank, which is preferai conically shaped, there -is disposed a drain valve through which fluid from line 40 flows through valve to line 44 and into steam seal tank 46 for a purpose be inafter set forth. An orifice aerator 48, of novel desi is situated through said tank and is fluidly connec by means of fluid pipe 50 to gas filter 52 having ther suitable filter means such as a fine glass wool (not shovt to filter the aerating gas passing therethrough. S rounding the filter 52 is a filter temperature controll jacket 54. The purpose of this jacket is to maintain gas filter at an elevated temperature at least at the d point of the aerating gas. The filter is operated with air passing down through the filter material in order eliminate pockets in the air system. All water va] passing through the filter is prevented from condens by the controlled temperature, thereby preventing c. densation of moisture which would flow by grai through the filter and carry contaminants to the ferm tor. The gas filter 52 at 56 is fluidly connected, at

option of the operator, to either aerating gas line 60 steam line 64 by means of valves 58 and 62 respective Any undesirable gas in the fermentor may be remo through lines 16 and 17 by means of a vacuum crea by steam jet 66 connected to steam line 64 throl valve 65. The mixture of gas and steam is exhaus into steam seal tank 46. During fermentation, the

tion of the jet 66 maintains a negative pressure of proximately 6 inches of water in the void above the c tents of the tank. This negative pressure aids in rring that any leakage which may occur in the head E the vessel remains in the system, thus reducing the azard to surrounding operating personnel. The steam :al tank 46 provides a means of isolating the system om other systems and from its environment. The steam :al tank is vented at 70 and has a drain valve 72 conected to steam line 40 in the manner similar to drain alve 30. Sewage line 74 acts as a drain for the steam :al tank.

Thermocouple 80, or other suitable device, is conected to the fermentation tank in order to determine re temperature of the tank which is maintained and con- 'olled by fermentor jacket 82 having suitable fluid lines 4 and 86.

The orifice aerator 48 shown in FIG. 2 is welded at n angle with the wall of tank 10 in order to assure rat any and all liquid in the gas line will remain within to fermentor. The assembly of the aerator is composed f three parts, an aerator barrel 88 of sufficient length be partially submerged in the contents of the tank, removable orifice plate 90 disposed in the end portion iereof which has an orifice $2. The orifice plate is 'ithdrawn or inserted through the upstream end of the erator barrel. It should be noted, however, that the )cation of the orifice in relation to the barrel 83 and the umber of the orifices used may vary according to the deired results, and it is within the skill of the operator 1 make a proper selection. The orifice diameter and the eration gas are such that the gas is forced through the rifice 92 at critical pressure ratio such that sonic gas elocities are produced. It has been found that a gas e.g. air) passing at sonic velocities through an orifice will reduce a large number of minute bubbles in the order I a greater than 25 and s less than .53 (critical pressure ratio of air C.) vhere A =area of orifice plate or barrel immediately refore the orifice; A =area of sharp edged orifice; =downstream pressure (absolute); P .=up-stream presure (absolute). It has been found that an orifice of b.0132 in diameter, an orifice plate diameter .156 in. ind a pressure difference across the orifice of Bit/in. vill pass air (at 15 C.) through the orifice at sonic 'elocities. However, it should be recognized that various :ombinations of diameters and pressures may be used as ong as they conform to the area and pressure ratios et forth. It has been determined that higher aeration :tficiencies may be obtained by use of additional orifices ind/or by introducing the gas tangentially to the tank to. The upstream end of the orifice aerator preferably ras positioned thereon a sampling adapter 24 similar to he sampling adapters 29 and 22 previously mentioned, he structure of which follows.

The sampling adapter is designed to allow easy access nto the interior of the tank 10 without the danger of :ontamination entering through the opening. The present :tructure accomplishes these results and is both simple ind efficient. FIG. 3 clearly shows the novel structure )f a body 94 and a suitable cap 96 having a washer 95 hereon enclosing a rubber diaphragm retained by a vasher 100 and a retaining ring 102. The rubber diahragm 98 is pierceaole, yet self-sealing and permits the :ntry and removal of instruments (3 g. hypodermic needle) to sample or inject materials in the tank. Noteworthy in this procedure is the omission of the use of any opening that would provide a source of contam nation. Previously known means for sampling did not incorporate the self-sealing, easy access to the contents of a tank. It should be stated that the cap 96 is optional and the rubber diaphragm may be of natural or synthetic rubber, or similar material that is pierceable and self-sealing. All the sampling adapters used are welded to their respective locations such as is shown in FIG. 3. These welds, where they extend to the interior of the tank, are ground and polished as previously mentioned.

The drain valves 30 and 72 are similar in construction and comprise a valve sealer 104 welded into a cooperating opening in the tank 10, and also being in fluid communication with fluid lines 49 and 44. Depending integrally from the valve sealer is a screw operated diaphragm valve comprising a diaphragm 105 made of a suitable flexible materal such as natural rubber, or a synthetic rubber such as neoprene. The diaphragm 105 is urged into engagement with the valve sealer at opening 106 to positively close this opening into tank 10. The means urging the diaphragm into the opening is not limited to the screw means 108, but rather any suitable means that will positively urge and maintain the diaphragm into the opening 106 could he used. When the screw 108 is operated by wheel 110 to urge the diaphragm 105 upwardly into opening 106 the contents of the tank 10 are restricted. However, it is the novel intention of this device to provide a valve sealer which is integral with the valve such that any seepage or undesirable leaking from around the diaphragm will not contaminate the environment of the tank. Previously known and used devices have attempted to assure a more secure shut off of the opening, which often times have resulted in complicated and time consuming closure devices. But regardless of how secure the closure is made, the danger exists that leaking may occur, and when pathogenic organisms are being processed, there is thus created a dangerous situation. Further, when this valve is used, sterilization is facilitated since there are no pipe stubs or other contaminant collectors existing that would prevent complete sterilization.

The valve sealer 104 transmits sealing fluid (e.g., steam) around the diaphragm from line 40 through to line 44 such that any material leaking from the diaphragm 1&5 is collected by the sealing fluid and transported to the steam seal tank 36. Thus, there is no loss of the contents of the tank 10 to the surroundings and consequently no danger to the operating personnel. From the steam seal tank 46 there is a sewage line 74. During operation of the fcrmentor, fluid is passed around the closed diaphragm and deposited into the steam seal tank 46. When the contents of the tank 10 are to be disposed of, the diaphragm is opened by means of wheel 110, permitting the contents of tank 10 to flow through opening 166 and pipe 44 into the steam seal tank 46 and thence becoming sewage upon the opening of drain valve 72. Fluid pipe 40 has a bypass line to the valve 72; which operates in the same safe manner as does valve 30.

Thus, it will be seen that at no time is there any substantial possibility of a loss of the contents of tank 10 to its environment.

The system is sterilized with steam as follows:

The media line 12 is connected to the fermentor 1t} and the valve 14 as well as the drain valve 39 are opened, while the vent 70 and the steam seal tank drain valve '72 are opened. Steam is introduced into tank 10 by its passage into the following from steam line 64: the filter jacket 54, through valve 62 and pipe 56 into gas filter 52, the media line 12, through valve 18 and line 16. When the temperature has reached a desired temperature, meas ured by thermocouple 80, the vent 70 is closed and the drain valve 36 is adjusted to allow condensate to drain, but to maintain pressure in the fermentor.

High pressure steam is introduced into the jacket 82 of the fermentor through line 84, and valve 65 to jet 66 is opened. The system is maintained at 250 F. for two hours.

After sterilization the system is cooled as follows:

The drain valve 72 is closed. All steam valves, except those to the air filter jacket, are closed and air is introduced into the fermentor to maintain pressure. Cool wateris circulated through the fermentor jacket 82, and subsequently the temperature is regulated to the desired level by means of the circulating fiuid through the jacket 82.

. After cooling, the system is prepared for innoculation:

Thefermentor drain valve 30 is closed and the fluid (e.g., steam) line 40 is opened at valve 42. The steam seal tank vent 70 is opened and the drain valve 72 is partially opened. Steam is introduced to the steam jet 66 and the supply of air is stopped. The desired volume of sterile medium is passed into the fermentor through the media line 12, inlet 15 and the valve 14. After passing the sterile media, the valve 14 is closed and the line 12 disconnected. The male portion of the coupling is capped and flooded with a liquid decontaminant. The medium is innurulated through the sampling adapter 20 with a hypodermic needle. A gas for aeration is introduced through orifice 92 into the culture at a controlled pressure producing sonic velocities that enhance growth of the microorganisms.

After completion of fermentation, the culture may be removed by way of the steam seal tank 46 as described above.

The system herein described has been used for studying the bio-engineering aspects of the fermentation processes of fastidious organisms. Cultures with characteristics similar to those prepared in laboratory equipment have been prepared in this system.

From the foregoing detailed description, it will be evident that there are a number of changes, adaptations and 6 modifications of the present invention that come with: the province of those skilled in the art; however, it intended that all such variations in structure, modes usage and materials be considered to be within the scor of the invention as limited solely by the appended claim We claim:

1. A process of culturing fastidious organisms con prising the removal of all projections from the interior 4 the tank, flushing the interior with a decontaminant mat rial before admitting a growth media, admitting tl growth media, maintaining an aerating gas filter mediu: heated. to above the dew point of aerating gas passir through said filter medium, aerating the growth med with said aerating gas at least at sonic velocities, provit ing a diaphragm drain valve in said tank, collecting ar transporting any seepage through said diaphragm dra valve by means of an enclosed fluid surrounding sa drain valve.

2. In a process of culturing organisms, in a valve tank, the improvement comprising collecting any seepa through a valve by means of an enclosed fluid in conta with said valve, and with the further improvement t aerating the organisms with a gas at least at sonic velo ities.

3. The process of claim 2 wherein the gas is pass through a filter heated to at least the dew point tempe ature of said gas.

4. The process of claim 3 wherein there is included tl step of smoothing all projections in the interior of ti said tank.

References Cited in the file of this patent UNITED STATES PATENTS 2,995,497 Heden Aug. 8, 19t 

1. A PROCESS OF CULTURING FASTIDIOUS ORGANISMS COMPRISING THE REMOVAL OF ALL PROJECTIONS FROM THE INTERIOR OF THE TANK, FLUSHING THE INTERIOR WITH A DECONTAMINANT MATERIAL BEFORE ADMITTING A GROWTH MEDIA, ADMITTING THE GROWTH MEDIA, MAINTAINING AN AERATING GAS FILTER MEDIUM HEATED TO ABOVE THE DEW POINT OF AERATING GAS PASSING THROUGH SAID FILTER MEDIUM, AREATING THE GROWTH MEDIA WITH SAID AERATING GAS AT LEAST AT SONIC VELOCITIES, PROVIDING A DIAPHRAGM DRAIN VALVE IN SAID TANK, COLLECTING AND TRANSPORTING ANY SEEPAGE THROUGH SAID DIAPHRAGM DRAIN VALVE BY MEANS OF AN ENCLOSED FLUID SURROUNDING SAID DRAIN VALVE. 